1. Field of the Invention
The invention relates to using proteolytically-deficient bacterial host strains. More particularly, the invention relates to such host strains that eliminate heterologous polypeptide degradation and improve yield of such polypeptides.
2. Description of Related Art
E. coli strains deficient in proteases or genes controlling the regulation of proteases are known. See, for example, Beckwith and Strauch, WO 88/05821 published Aug. 11, 1988; Chaudhury and Smith, J. Bacteriol., 160: 788-791 (1984); Elish et al., J. Gen. Microbiol., 134: 1355-1364 (1988); Baneyx and Georgiou, xe2x80x9cExpression of proteolytically sensitive polypeptides in Escherichia coli,xe2x80x9d In: Stability of Protein Pharmaceuticals, Vol. 3: Chemical and Physical Pathways of Protein Degradation, Ahem and Manning, eds. (Plenum Press, New York, 1992), p. 69-108.
Some of these strains have been used in attempts to produce efficiently proteolytically sensitive proteins, particularly those of potential medical or commercial importance. U.S. Pat. No. 5,508,192 (to Georgiou et al.) describes the construction of many protease-deficient and/or heat-shock protein-deficient bacterial hosts. Such hosts include single-, double-, triple-, or quadruple-protease-deficient bacteria and single-protease bacteria that also carry a mutation in the rpoH gene. Examples of the protease-deficient strains disclosed include those omitting degP, ompT, ptr3, and/or prc (tsp), and a degP rpoH strain reported to produce large titers of recombinant proteins in E. coli. Park et al., Biotechnol. Prog., 15: 164-167 (1999) also reported that a strain (HM114) deficient in two cell-envelope proteases (degP, prc) grew slightly faster and produced more fusion protein than the other strains deficient in more proteases. They claimed that this strain grew to a cell dry weight of 47.86 g/L in 29 hours using pH-stat, fed-batch cultivation. The protein produced was protein A-xcex2-lactamase fusion protein, which gave 30% higher xcex2-lactamase activity than that obtained from its parent strain KS272.
The Prc protein was first isolated by Ham et al., J. Bacteriol 173: 4799-4813 (1991) as the periplasmic protease that cleaves the carboxyl-terminus of periplasmic penicillin binding protein 3 (PBP3). Subsequently, it was also identified as a protease that selectively degrades proteins with a non-polar C-terminus and was re-named Tsp (Silber et al., Proc. Natl. Acad. Sci. USA, 89: 295-299 (1992)). The prc gene was shown to encode a 75-kDa protein, which is required for protection of cells from combined thermal and osmotic stress (Hara et al., supra). It has been confirmed that the C-terminal sequences determine the substrate preference (Keiler et al., Protein Sci., 4: 1507-1515 (1995)). The amount of cleavage is sensitive to the identity of residues or functional groups at the C-terminus of the substrate protein. The presence of a free xcex1-carboxyl group is important in determining whether closely related peptides with non-polar C-terminal sequences are cleaved efficiently by Prc.
Prc homologs have been identified in a divergent group of prokaryotes, including several cyanobacteria (Brand et al., Plant Mol. Bio., 20: 481-491 (1992); Shestakov et al., J. Biol. Chem., 269: 19354-19359 (1994)), Neisseria gonorrhoeae (Black et al., J. Bacteriol., 177: 1952-1958 (1995)), Haemophilus influenzae (Fleischmann et al., Science, 269: 496-512 (1995)), and Bartonella bacilliformis (GenBank accession no. L37094). A domain in the Prc family of proteins is similar to a domain in the retinol-binding proteins, indicating a common folding domain that may form a binding pocket in these proteins for hydrophobic substrates (Silber et al., supra; Shestakov et al., supra).
Hara et al., supra, discovered that the thermoresistant revertants of xcex94prc mutants contain extragenic suppressor (spr) mutations. They further identified the wild-type spr gene product to be a lipoprotein in the envelope fraction. They suspected that the wild-type spr gene could be a peptidoglycan-hydrolyzing enzyme (Hara et al, Microbial Drug Resistance, 2: 63-72 (1996)). When the spr is not functional in a prc-plus background, a suppressor for spr mutation was identified to be PBP7, another penicillin-binding protein (Hara et al., 1996, supra). The cloning of spr and the preparation of a xcex94prc mutant in which Spr is not degraded by the protease are also described in Hara et al., Abstract for Table Ronde Roussel Uclat no. 86, Versailles, May 1997, where the authors concluded that prc and spr are mutual suppressors.
Three multicopy prc suppressors have also been isolated using the conditional lethal phenotype of a prc (tsp) null strain of E. coli (Bass et al., J. Bacteriol., 178: 1154-1161 (1996)). None of them relate to the spr gene. One set of these suppressors is two putative protease genes in tandem that map to 72.5 min on the chromosome. These two genes are htrA homologs, which encode proteins that are 58 and 35% identical, respectively, to the HtrA (DegP) serine protease. Another type of suppressor identified is the dksA (dnak suppressor) gene, which is also a multicopy suppressor of defects in the heat-shock genes dank, dnaj and grpE. The dksA gene was also independently isolated as a multicopy suppressor of a mukB mutation, which is required for chromosomal partitioning. The third type is a truncated lipoprotein A (rlpA) gene.
The gene degP appears to control synthesis of a cell-envelope protease DegP (HtrA). A degP-deficient mutant was first constructed and recombined into an E. coli chromosome by Beckwith and Strauch, supra. HtrA has a high molecular mass of about 500 kDa, which is a heat-shock protein whose proteolytic activity is essential for the survival of E. coli at high temperatures such as above 42xc2x0 C. (Skorko-Glonek et al., Gene, 163: 47-52 (1995)). A number of ordinarily unstable cell-envelope proteins can be stabilized by the degP mutation (Strauch and Beckwith, Proc. Natl. Acad. Sci. USA, 85: 1676-1580 (1988)). Recently, HtrA protein was reported to behave as a dodecamer consisting of two stacks of hexameric rings by electron microscopy and chemical cross-linking analysis (Kim et al., J. Mol. Biol., 294: 1363-1374 (1999)). Unfolding of protein substrates, such as by exposure to high temperature or reduction of disulfide bonds, is essential for their access into the inner chamber of the double ring-shaped HtrA, where cleavage of peptide bonds may occur (Kim et al., supra).
Many heterologous polypeptides have been produced in various strains deficient in proteases.
However, many of the strains gave relatively low product titer and/or poor growth. There is a need to provide a bacterial strain deficient in proteases that does not result in clipping of the product and provides high product titer.
Accordingly, the present invention is as claimed. In one aspect the present invention provides E. coli strains that are deficient in chromosomal degP and prc encoding protease DegP and Prc, respectively, and harbor or comprise a mutant spr gene the product of which gene suppresses growth phenotypes exhibited by strains harboring prc mutants. Preferably the strain is not deficient in chromosomal ptr3 encoding Protease III and/or in chromosomal ompT encoding protease OmpT. Preferably, the E. coli strain is engineered by introducing the mutant spr gene to a degPxcex94 prcxcex94 strain for survival in the stationery phase of a high-cell density E. coli fermentation process.
In another embodiment, the strain comprises nucleic acid encoding a polypeptide heterologous to the strain, preferably a proteolytically-sensitive polypeptide, and more preferably a eukaryotic polypeptide.
In a further embodiment, the invention provides a method for producing a heterologous polypeptide, i.e., one that is heterologous to the strain. This method comprises first culturing an E. coli strain that is deficient in chromosomal prc encoding protease Prc and harbors or comprises a mutant spr gene the product of which gene suppresses growth phenotypes exhibited by strains harboring prc mutants. This strain also comprises nucleic acid encoding the heterologous polypeptide. The culturing is such that the nucleic acid is expressed. In a second step of this method, the polypeptide is recovered from the strain, whether from the cytoplasm, periplasm, or culture medium, preferably the periplasm or culture medium, and most preferably from fermentation whole broth. Preferably, the polypeptide is Apo2 ligand or an antibody, including an antibody fragment.